Polysaccharide conjugate vaccines are proving to be an effective means to generate protective immune responses so as to prevent a wide range of diseases. The traditional chemical producing approach has enabled the production of several highly successful conjugated vaccines in clinical use. However, the traditional approach suffers from multiple fermentations, purification steps, low yields and non-specific chemical conjugation, thus leading to high costs for vaccine production. The objective of this application is to explore a recently established bacterial protein O-glycosylation system to obtain polysaccharide conjugate vaccines in a facile, efficient, and easily applicable manner. This bacterial glycosylation system includes a highly promiscuous protein, PglL, which catalyzes the transfer of polysaccharides from a diphospho-lipid donor to target proteins. The proof-of-concept experiments led compelling evidence for further exploration of such a system in conjugate vaccine development. Furthermore, the success of this proposed approach also heavily relies on the in-depth understanding of polysaccharide biosynthesis in bacteria, which we have been studying in the past several years with a combination of genetic, chemical, and biochemical approaches. Phase I of this STTR application will demonstrate the feasibility of this approach using a pneumococcal pathogen (Streptococcus pneumoniae serotype 14) as a model. We plan to focus our efforts on the following two specific aims: 1. We will establish an in vitro reaction system based on the chemical synthesis of lipid pyrophosphate linked monosaccharides and enzymatic assembly of the corresponding CPS repeating unit. Besides confirmation of PglL activity towards this specific CPS (CPS14), the peptide preference in the context of both the primary sequence and local secondary structure of proteins will be explored to obtain the most favorable substrate sequence for PglL. Finally, a commonly used carrier protein of polysaccharide conjugate vaccines (CRM197) will be engineered to serve as the acceptor substrate for an in vivo production system. 2. We aim to achieve an E. coli strain producing CRM197 conjugated CPS14. E. coli K12 W3110 will be utilized as a host strain. Two genes (wecA: initial glycosyltransfer reaction, waaL: ligation of nascent polysaccharide chains to the core-Lipid A) which are vital to the LPS biosynthetic pathway and may interfere with PglL O-glycosylation will be disrupted. The feasibility of heterologously expressing of CPS14 in the host strain will then be demonstrated. Genes encoding PglL and the favorable CRM197 variants, along with the CPS14 gene cluster required for the heterologous expression of CPS14, will be introduced into the mutant host strain. Expression, purification, characterization and bioassays of CPS14-CRM197 conjugates from fermentation of this engineered strain will be explored. Successful demonstration of the production of CRM197 conjugated CPS14 will set the groundwork for producing CPS conjugates from other S. pneumoniae serotypes as well as polysaccharides from other pathogens. Given that the CPS biosynthetic gene clusters of 90 S. pneumoniae serotypes have been completely sequenced, the accessibility of polysaccharide biosynthesis loci will enable generalization of our approach for the production of a variety of polysaccharide conjugate vaccines. PUBLIC HEALTH RELEVANCE: Bacterial infections are one of the major health problems worldwide. Polysaccharides, forming a thick capsule that surrounds the bacterial pathogen, represent a major determinant of pathogenicity. With the increasing emergence of resistance toward major antibiotics, development of polysaccharide-based vaccines provides an attractive approach for fighting the infectious diseases. Polysaccharides are better to be conjugated to a carrier protein as conjugate vaccines to enhance their efficacy. The traditional chemical approach of polysaccharide conjugate vaccine production has enabled the production of several highly successful conjugated vaccines currently in clinical use. However, the traditional method suffers from complex production steps, low yields and impure products, thus leading to high costs for vaccine production. The objective of this application is to develop a novel method for polysaccharide conjugate vaccine production. With this method, we can obtain polysaccharide conjugate vaccines in a facile, efficient, and easily applicable manner. This method will firstly be explored with a single pathogen as a model. Then the established method can be easily applied to other pathogens.